WO1995005471A2 - Method for stable transformation of plants - Google Patents
Method for stable transformation of plants Download PDFInfo
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- WO1995005471A2 WO1995005471A2 PCT/EP1994/002566 EP9402566W WO9505471A2 WO 1995005471 A2 WO1995005471 A2 WO 1995005471A2 EP 9402566 W EP9402566 W EP 9402566W WO 9505471 A2 WO9505471 A2 WO 9505471A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
Definitions
- the said substrate involves not the whole but only part of the T-DNA border sequence, it is to be ensured that the said partial sequence still comprises those parts of the T-DNA border sequence that encompasses the recognition and cleavage site of the VirD2 protein.
- the chimeric recombinant DNA construct as described above is preferably a single stranded DNA construct. Also comprised within the scope of the invention is a double-standed molecule with a single-stranded overhang which is a substrate for VirD2 or a chimeric recombinant DNA construct negatively supercoiled (form I) containing border sequences as the preferred substrate for NirDl/VirD2 catalyzed cleavage.
- the said VirD2 protein Upon in vitro cleavage of the T-D ⁇ A border sequence or of functional parts thereof, using the VirD2 protein, the said VirD2 protein remains covalently attached to the cleaved D ⁇ A forming the D ⁇ A/protein complex according to the invention.
- the present invention thus further relates to a method of preparing a D ⁇ A/protein complex as described before, comprising:
- step (b) in vitro cleaving of the D ⁇ A substrate prepared according to step (a) by means of VirD2 protein, which may be accompanied by further Nir proteins such as, for example VirDl and/or VirE2 and/or any other D ⁇ A binding protein, which is able to protect the D ⁇ A from nuclease attack.
- VirD2 protein which may be accompanied by further Nir proteins such as, for example VirDl and/or VirE2 and/or any other D ⁇ A binding protein, which is able to protect the D ⁇ A from nuclease attack.
- the substrate to be used in the VirD2 cleavage reaction may contain one or more T-D ⁇ A border sequences or one or more functional parts of T-D ⁇ A border sequences, which may be of the same or of different specificities. This means, that the substrate may involve one or more D ⁇ A sequences with left border specificity or one or more D ⁇ A sequences with right border specificity or combinations of D ⁇ A sequences with left and right border specificities.
- the substrate may involve further D ⁇ A sequences such as, for example, overdrive sequences. - 1
- the present invention is in the field of plant genetic engineering.
- it relates to a method for producing stably transformed plant material using a specifically designed DNA/protein complex.
- the invention further relates to the said DNA protein complex itself and to the plant material transformed therewith.
- the Agrobacteriwn plant transformation system is widely used for the stable transformation of higher plants.
- genes to be transferred are carried by the T-DNA, a well-defined region of the Agrobacteriwn Ti plasmid.
- the Ti plasmid also contains a virulence (vir) region, which encodes proteins involved in the transformation via Agrobacteriwn of plant cells. At least one of these proteins, NirD2 is involved in targeting to the plant nucleus and integration into the plant genome [Tinland et al, Proc ⁇ atl Acad Sci USA 89: 7442-7446, 1992; Mayerhofer et al, EMBO J 10: 697-704 (1991)].
- the main object of the invention to provide a method for producing stably transformed plant material, including phenotypically normal looking and preferably fertile plants, which method does not involve Agrobacteriwn transformation.
- a specifically adapted D ⁇ A/protein complex comprising a chimeric recombinant D ⁇ A, which may comprise, for example, an expressible D ⁇ A operably linked to suitable plant expression signals involving promoter and termination sequences and covalently associated therewith a VirD2 protein.
- This D ⁇ A/protein complex may be obained by first providing a recombinant D ⁇ A construct that comprises in operable linkage to the elements already mentioned above at least one T-D ⁇ A border sequence or functional parts thereof as a substrate in the VirD2
- DNA substrates comprising either a left or a right border element or functional parts thereof or a combination of left and right border elements or of functional parts thereof, that in addition may be accompanied by one or more overdrive sequences.
- substrates comprising at least two T-DNA border elements of the same specificity, that is right or left border elements that in addition may be accompanied by one or more overdrive sequences.
- the DNA substrates according to the invention preferably involve at least those parts obtainable from T-DNA border sequences that comprise in addition to the cleavage site at least the following core sequence according to SEQ ID NO 1:
- K being either guanosine or thymidine/uridine; but more preferably the core sequence according to SEQ ID NO 2:
- the D ⁇ A substrate involves a D ⁇ A sequence that comprises between 6 and approximately 50 nucleotides, preferably between 10 and approximately 40 nucleotides and which can be described by the following general formula according to SEQ ID NO 3:
- N may be any of the nucleotides selected from the group consisting of adenosine, guanosine, cytidine and tymidine or uridine
- K is either guanosine or thymidine/uridine
- n is an integer between 0 and 42, preferably between 0 and 25, more preferably between 0 and 14
- m is an integer between 2 and 10, preferably between 3 and 10, more preferably between 4 and 8 and wherein the wedge indicates the position of the cleavage site.
- the DNA substrate involves a DNA sequence that comprises between 10 and approximately 50 nucleotides, but preferably between 14 and 29 nucleotides and which can be described by the following general formula according to SEQ ID NO 5:
- N may be any of the nucleotides selected from the group consisting of adenosine, guanosine, cytidine or thymidine/uridine; B is guanosine or cytidine or tymidine/uridine; H is adenosine or cytidine or tymidine/uridine; M is adenosine or cytidine; R is guanosine or adenosine; Y is tymidine/uridine or cytidine; K is either guanosine or thymidine/uridine; and n is an integer between 0 and 40, but preferably between 2 and 17; and m is an integer between 2 and 10, preferably between 3 and 10, more preferably between 4 and 8 and wherein the wedge indicates the position of the cleavage site.
- DNA substrate involving a DNA sequence that comprises between 17 and 25 nucleotides and which can be described by the following general formula according to SEQ ID NO 6:
- N may be any of the nucleotides selected from the group consisting of adenosine, guanosine, cytidine and thymidine/uridine; B is guanosine or cytidine or tymidine/uridine; H is adenosine or cytidine or tymidine/uridine; M is adenosine or cytidine; R is guanosine or adenosine; Y is tymidine/uridine or cytidine; and n is an integer between 0 and 12, preferably between 0 and 8; including any DNA sequences that are structurally and/or functionally homologue thereto.
- substantially sequence homology means close structural relationship between sequences of nucleotides.
- substantially homologous DNA sequences may be 60 % homologous, preferably 80 % and most preferably 90 % or 95 % homologous, or more.
- Homology also includes a relationship wherein one or several subsequences of nucleotides or amino acids are missing, or subsequences with additional nucleotides or amino acids are interdispersed.
- the transformation frequency and also the quality of the integrated DNA can be improved considerably. This is especially true with regard to stable transformation events, which occur more frequently as compared to conventional, non-protein associated DNA constructs.
- the present invention thus further comprises a method of transforming plant material comprising
- step (b) in vitro cleaving of the DNA substrate prepared according to step (a) by means of VirD2 protein, which may be accompanied by further Vir proteins such as, for example VirDl and/or VirE2 and/or any other DNA binding protein, which is able to protect the DNA from nuclease attack;
- the DNA/VirD2 protein complex according to the invention may be accompanied by further Vir proteins, [which, for example, are capable of catalyzing physical changes in the complex] such as, for example, VirE2, which is known to bind to ssDNA, and/or VirDl.
- VirE2 can be purified by methods known in the art such as those described in Christie et al [J Bacteriol 170(6): 2659-2667 (1988)].
- the purificatiion of the VirDl protein can be achieved according to the method provided in the following examples.
- VirD2 protein and optionally further Vir proteins that may be involved in the cleavage reaction, in amounts and in a purity that is suitable for carrying out the method according to the invention. This can be achieved by overproducing the Vir proteins in a suitable host organism and purifying them in a multiple-step procedure.
- the main object of the present invention is a DNA/protein complex comprising operably linked to an expressible DNA at least one T-DNA border sequence or functional parts thereof and covalently associated therewith a VirD2 protein, which complex can be suitably used in a process for transforming DNA into plant material.
- the DNA/VirD2 protein complex may contain non-covalently associated further Vir proteins such as, for example, VirDl and/or VirE2.
- the DNA to be used in the process according to the invention for transforming plant material may be either of homologous or heterologous origin with respect to the plant material involved or it may be of synthetic origin or both.
- the coding DNA sequence can be constructed exclusively from genomic DNA, from cDNA or from synthetic DNA. Another possibility is the construction of a hybrid DNA sequence consisting of both cDNA and genomic DNA and/or synthetic DNA.
- the cDNA may originate from the same gene as the genomic DNA, or alternatively both the cDNA and the genomic DNA may originate from different genes. In any case, however, both the genomic DNA and/or the cDNA may each be prepared individually from the same or from different genes.
- Synthetic DNA is to be understood as comprising DNA sequences that have been been prepared entirely or at least partially by chemical means. Synthetic DNA sequences may be suitably used, for example, for modifying native DNA sequences in terms of codon usage, expression efficiency, etc. Examples of synthetic genes include the PAT gene and the endotoxin genes of Bacillus thuringiensis.
- DNA sequence to be transformed into the recipient plant material contains portions of more than one gene, these genes may originate from one and the same organism, from several organisms that belong to more than one strain, one variety or one species of the same genus, or from organisms that belong to more than one genus of the same or of another taxonomic unit (kingdom).
- these genes may originate from one and the same organism, from several organisms that belong to more than one strain, one variety or one species of the same genus, or from organisms that belong to more than one genus of the same or of another taxonomic unit (kingdom).
- Chimaeric recombinant DNA molecules that comprise an expressible DNA, but especially a structural gene, preferably a heterologous structural gene operably linked with expression signals active in plant cells, such as promoter and termination sequences, as well as, optionally, with further coding and/or non-coding sequences of the 5' and/or 3' region may also be preferably used within the transformation process as part of the DNA/protein complex according to the present invention.
- Especially suitable for use in the process according to the invention are all those structural genes which upon expression lead to a protective effect in the transformed plant cells, also in the tissues developing therefrom and especially in the regenerated plants, for example increased resistance to pathogens (for example to phytopathogenic fungi, bacteria, viruses, etc.); resistance to chemicals [for example to herbicides (e.g. triazines, sulfonylureas, imidazolinones, triazole pyrimidines, bialaphos, glyphosate, etc.), insecticides or other biocides]; resistance to adverse environmental factors (for example to heat, cold, wind, adverse soil conditions, moisture, dryness, etc.).
- pathogens for example to phytopathogenic fungi, bacteria, viruses, etc.
- chemicals for example to herbicides (e.g. triazines, sulfonylureas, imidazolinones, triazole pyrimidines, bialaphos, glyphosate,
- Resistance to insects can be conferred, for example, by a gene coding for a polypeptide that is toxic to insects and/or their larvae, for example the crystalline protein of Bacillus thuringiensis [B.t.].
- B.t. Bacillus thuringiensis
- synthetic B.t. genes such as those disclosed in, for example, Koziel M.G. et al, Bio Technology U: 194-200 (1993).
- a second class of proteins mediating resistance to insects comprises the protease inhibitors.
- Protease inhibitors are a normal constituent of plant storage structures and are therefore normally located in vacuoles or protein bodies. It has been demonstrated that a Bowman-Birk protease inhibitor isolated from soybeans and purified inhibits the intestinal protease of Tenebrio larvae. The gene that codes for the trypsin inhibitor from the cowpea is described in Hilder et al (1987).
- insects for example, have a cuticular skeleton in which chitin micelles in lamellar layers are embedded in a base substance.
- a great many phytopathogenic fungi also contain chitin as an integral part of their hypha and spore structures, for example Basidiomycetes (smut and rust fungi), Ascomycetes and Fungi imperfecti (including Alter na ⁇ a and Bipolar is, Exerophilum turcicum, Colletotricum, Gleocercospora and Cercospora).
- Chitinase is capable of inhibiting the mycelial growth of certain pathogens both in vitro and in vivo.
- a plant organ or tissue that is capable of expressing chitinase constitutively or in response to the penetration of a pathogen can therefore protect itself from attack by a large number of different fungi.
- a further gene, which encodes an enzyme which presumably plays a central role in the plant's defence mechanism against pathogens is the ⁇ -l,3-glucanase gene, that may thus also be used for protecting plants against a fungal attack, alone or in combination with a chitinase gene.
- a further class of genes that may be used within the scope ot this invention are those coding for the so-called lyric peptides. These are natural or synthetic peptides having anti-pathogenic activity which are capable of penetrating, lysing or otherwise damaging the cell membrane of pathogens.
- lytic peptides that may be used within the scope of the present invention are known both from animal sources [including insects] and from plant and microbial sources and include, for example, the defensins, cecropins, thionins and mellitins of mammals, and the defensins, magainins, attacins, dipterins, sapecins, caerulins and xenopsins of insects, and hybrids thereof.
- the amino acid sequences of various lytic peptides are shown in the following publications: WO 89/11291; WO 86/04356; WO 88/05826; US 4,810,777; WO 89/04371.
- Lytic peptides in the broadest sense of the term are also to be understood as being compounds whose ability to penetrate, lyse or damage cell membranes is based on enzymatic activity, for example lysozymes and phospholipases.
- lytic peptides especially being suitable for the latter purpose, in conjunction with the auxiliaries and/or additives customarily used for this purpose.
- genes that may be used in the scope of the present invention are those coding for phospholipid transfer proteins disclosed, for example, in WO 92/20801.
- a further class of genes that may be used within the scope of the present invention comprises genes which encode pathogenesis-related proteins [PRPs] such as PR-1A, PR-IB, PR-1C, PR-R major, PR-R minor, PR-P, PR-Q, PR-2, PR-2 ⁇ PR-2", PR-N, PR-O, PR-O', PR-4, SAR8.2a-e, cucumber chitinase/lysozyme, cucumber basic peroxidase, tobacco basic glucanase and tobacco basic chitinase/lysozyme, tobacco acidic chitinase/lysozyme.
- PRPs pathogenesis-related proteins
- the DNA sequence according to the invention can also be used for the production of desirable and useful compounds in the plant cell as such or as part of a unit of higher organisation, for example a tissue, callus, organ, embryo or a whole plant.
- Genes that may also be used within the scope of the present invention include, for example, those which lead to increased or decreased formation of reserve or stored substances in leaves, seeds, tubers, roots, stems, etc. or in the protein bodies of seeds.
- the desirable substances that can be produced by transgenic plants include, for example, proteins, carbohydrates, amino acids, vitamins, alkaloids, flavins, perfumes, colourings, fats, etc..
- DNA sequence according to the invention may also be associated with the DNA sequence according to the invention structural genes that code for pharmaceutically acceptable active substances, for example hormones, immunomodulators and other physiologically active substances.
- genes that can come into consideration within the scope of this invention therefore include, but are not limited to, for example, plant-specific genes, such as the zein gene from maize, the avenin gene from oats, the glutelin gene from rice, etc., mammal-specific genes, such as the insulin gene, the somatostatin gene, the interleukin genes, the t-PA gene, etc., or genes of microbial origin, such as the NPT II gene, etc. and synthetic genes, such as the insulin gene, etc..
- plant-specific genes such as the zein gene from maize, the avenin gene from oats, the glutelin gene from rice, etc.
- mammal-specific genes such as the insulin gene, the somatostatin gene, the interleukin genes, the t-PA gene, etc.
- genes of microbial origin such as the NPT II gene, etc.
- synthetic genes such as the insulin gene, etc.
- genes that code for a useful and desirable property within the scope of this invention it is also possible to use genes that have been modified previously in a specific manner using chemical or genetic engineering methods.
- the broad concept of the present invention also includes genes that are produced entirely or patially by chemical synthesis. Genes or DNA sequences that may be used within the scope of the present invention are therefore both homologous and heterologous gene(s) or DNA and also synthetic gene(s) or DNA according to the definition given within the scope of the present invention.
- the insulin gene may be mentioned at this point as an example of a synthetic gene.
- the coding gene sequences first to be linked in operable manner to expression sequences capable of functioning in plant cells.
- hybrid gene constructions within the scope of the present invention therefore comprise, in addition to the DNA sequence according to the invention, one or more struc ⁇ tural gene(s) and, in operable linkage therewith, expression signals which include both promoter and terminator sequences and other regulatory sequences of the 3' and 5' untranslated regions.
- Any promoter and any terminator capable of bringing about an induction of the expression of a coding DNA sequence may be used as a constituent of the hybrid gene sequence.
- the said expression signals may promote continuous and stable expression of the gene.
- expression signals originating from genes of plants or plant viruses are those of the Cauli ⁇ flower Mosaic Virus genes (CaMV) or homologous DNA sequences that still have the characteristic properties of the mentioned expression signals.
- bacterial expression signals especially the expression signals of the nopaline synthase genes (nos) or the opine synthase genes (ocs) from the Ti-plasmids of Agrobacteriwn tumefaciens.
- ubiquitine promoters actin promoters, historie promoters and tubulin promoters.
- 35S and 19S expression signals of the CaMV genome or their homologues which can be isolated from the said genome using molecular biological methods, as described, for example, in Maniatis et al (1982), and linked to the coding DNA sequence.
- homologues of the 35S and 19S expression signals are * to be understood as being sequences that, despite slight sequence differences, are substantially- homologous to the starting sequences and still fulfill the same function as those starting sequences.
- the expression signals may also comprise tissue-preferential or tissue specific promoters.
- tissue-preferential promoter is used to indicate that a given expression signal will promote a higher level of transcription of an associated expressible DNA, or of expression of the product the said DNA as indicated by any conventional RNA or protein assay, or that a given DNA sequence will demonstrate some differential effect; i.e., that the transcription of the associated DNA sequences or the expression of a gene product is greater in some tissue than in all other tissues of the plant.
- the tissue- preferential promoter may direct higher expression of an associated gene product in leaves, stems, roots and/or pollen than in seed.
- a tissue-preferential promoter which may be suitably used within the scope of the present invention, is a pith-preferred promoter isolated from a maize TrpA gene.
- tissue-specific promoter is used to indicate that a given regulatory DNA sequences will promote transcription of an associated expressible DNA sequence entirely in one or more tissues of a plant, or in one type of tissue, e.g. green tissue, while essentially no transcription of that associated coding DNA seuquence will occur in all other tissues or types of tissues of the plant.
- Numerous promoters whose expression are known to vary in a tissue specific manner are known in the art.
- One such example is the maize phosphenol pyruvate carboxylase [PEPC], which is green tissue-specific [Hudspeth RL and Grula JW, 1989].
- Other green tissue-specific promoters include chlorophyll a/b binding protein promoters and RUBISCO small subunit promoters.
- pollen-specific promoters such as those obtainable from a plant calcium-dependent phosphate kinase [CDPK]) gene.
- a developmentally regulated promoter can also be used.
- any promoter which is functional in the desired host plant can be used to direct the expression of an associated gene.
- leader sequence between the promoter sequence and the adjacent coding DNA sequence, the length of the leader sequence being so selected that the distance between the promoter and the DNA sequence according to the invention is the optimum distance for expression of the associated structural gene.
- chimaeric genes include, for example, sequences that are capable of regulating the transcription of an associated DNA sequence in plant tissues in the sense of induction or repression.
- Another class of genes that are inducible in plants comprises the light-regulated genes, especially the nuclear-coded gene of the small subunit of ribulose-l,5-biphosphate carboxylase (RUBISCO).
- RUBISCO ribulose-l,5-biphosphate carboxylase
- Morelli et al, Nature 315: 200-204 (1985) have shown that the 5 '-flanking sequence of a RUBISCO gene from the pea is capable of transferring light-inducibility to a reporter gene, provided the latter is linked in chimaeric form to that sequence. It has also been possible to extend this observation to other light-induced genes, for example the chlorophyll-a/b-binding protein.
- a further group of regulatable DNA sequences comprises chemically regulatable sequences that are present, for example, in the PR (pathogenesis-related) protein genes of tobacco and are inducible by means of chemical regulators such as those described in EP-A 332,104.
- the regulatable DNA sequences mentioned by way of example above may be of both natural and synthetic origin, or they may comprise a mixture of natural and synthetic DNA sequences.
- a chimeric recombinant DNA construct comprising an expressible DNA as described above involving, for example, one or more protein encoding DNA sequences, promoter and termination sequences and optionally further regulatory sequences of the 3' and 5' untranslated regions, is covalently associated with a VirD2 protein.
- the DNA/protein complex according to the invention can be introduced into the plant cell in a number of ways that are well known to those of skill in the art.
- methods of transforming plant cells include microinjection [Crossway et al, BioTechniques 4: 320-334 (1986); Neuhaus et al, Theor Appl Genet 75, 30-36 (1987)], electroporation [Riggs et al, Proc Nat Acad Sci USA 83. 5602-5606 (1986)], direct gene transfer [Paszkowski et al, EMBO J.
- Possible methods for the direct transfer of the DNA/protein complex according to the invention into a plant cell comprise, for example, the treatment of protoplasts using procedures that modify the plasma membrane, for example, polyethylene glycol treatment, heat shock treatment or electroporation, or a combination of those procedures [Shillito et al, Bio Technology, 3: 1099-1103 (1985)].
- plant protoplasts together with the DNA protein complex according to the invention are subjected to electrical pulses of high field strength. This results in a reversible increase in the permeability of biomembranes and thus allows the insertion of the DNA/protein complex according to the invention.
- Electroporated plant protoplasts renew their cell wall, divide and form callus tissue. Selection of the transformed plant cells can take place with the aid of the above-described phenotypic markers.
- Co-transformation is a method that is based on the simultaneous taking up and integration of various DNA molecules (non-selectable and selectable genes) into the plant genome and that therefore allows the detection of cells that have been transformed with non-selectable genes.
- Further means for inserting the DNA/protein complex according to the invention directly into a plant cell comprise using purely physical procedures, for example by microinjection using finely drawn micropipettes [Neuhaus et al (1987)] or by bombarding the cells with microprojectiles that are coated with the transforming DNA ["Microprojectile Bombardment”; Wang Y-C et al, Plant Mol. Biol. ⁇ : 433-439 (1988)] or are accelerated through a DNA containing solution in the direction of the cells to be transformed by a pressure impact thereby being finely atomized into a fog with the solution as a result of the pressure impact [EP-A-434,616].
- Microprojectile bombardment has been advanced as an effective transformation technique for cells, including cells of plants.
- Sanford et al (1987) it was reported that microprojectile bombardment was effective to deliver nucleic acid into the cytoplasm of plant cells of Allium cepa (onion).
- Christou et al (1988) reported the stable transformation of soybean callus with a kanamycin resistance gene via microprojectile bombardment.
- Christou et al reported penetration at approximately 0.1 % to 5 % of cells.
- Christou further reported observable levels of NPTII enzyme activity and resistance in the transformed calli of up to 400 mg/1 of kanamycin.
- McCabe et al (1988) report the stable transformation of Glycine max (soybean) using microprojectile bombardment. McCabe et al further report the recovery of a transformed Rj plant from an R Q chimaeric plant.
- the present invention therefore also comprises transgenic plant material, selected from the group consisting of protoplasts, cells, calli, tissues, organs, seeds, embryos, ovules, zygotes, etc. and especially, whole and preferably phenotypically normal plants, that has been transformed by means of the processes described above and comprises the recombinant DNA according to the invention in expressible form, and processes for the production of the said transgenic plant material.
- transgenic plant material selected from the group consisting of protoplasts, cells, calli, tissues, organs, seeds, embryos, ovules, zygotes, etc. and especially, whole and preferably phenotypically normal plants, that has been transformed by means of the processes described above and comprises the recombinant DNA according to the invention in expressible form, and processes for the production of the said transgenic plant material.
- Transformation of the plant cells includes separating transformed cells from those that have not been transformed.
- One convenient method for such separation or selection is to incorporate into the material to be inserted into the transformed cell a gene for a selection marker.
- the translation product of the marker gene will then confer a phenotypic trait that will make selection possible.
- the phenotypic trait is the ability to survive in the presence of some chemical agent, such as an antibiotic, e.g., kanamycin, G418, paromomycin, etc., which is placed in a selection media.
- genes that confer antibiotic resistance include those coding for neomycin phosphotransferase kanamycin resistance, [Nelten et al, EMBO J. 3: 2723-2730 (1984)]; hygromycin phosphotransferase (hygromycin resistance, [van den Elzen et al, Plant Molecular Biology 5: 299-392 (1985)], the kanamycin resistance ( ⁇ PT II) gene derived from Tn5 Bevan et al, Nature 304: 184-187 (1983); [McBride et al, Plant Molecular Biology 14: 266-276 (1990)], the PAT gene described in Thompson et al, EMBO J 6: 2519-2523 (1987), and chloramphenicol acetyltransferase.
- GUS beta-glucuronidase
- surviving cells are selected for further study and manipulation. Selection methods and materials are well known to those of skill in the art, allowing one to choose surviving cells with a high degree of predictability that the chosen cells will have been successfully transformed with exogenous DNA.
- Positive clones are regenerated following procedures well-known in the art. Subsequently transformed plants are evaluated for the presence of the desired properties and/or the extent to which the desired properties are expressed.
- a first evaluation may include, for example, the level of bacterial/fungal resistance of the transformed plants, stable heritability of the desired properties, field trials and the like.
- transgenic plants in particular transgenic fertile plants transformed by means of the aforedescribed process of the invention and their asexual and/or sexual progeny, which still display the new and desirable property or properties due to the transformation of the mother plant.
- the transgenic plant according to the invention may be a dicotyledonous or a monocotyledonous plant.
- Preferred are monocotyledonous plants of the Graminaceae family involving Lolium, Zea, Triticum, Triticale, Sorghum, Saccharum, Bromus, Oryzae, A vena, Hordeum, Secale and Setaria plants.
- transgenic maize, wheat and barley plants are especially preferred.
- rape seed and sunflower are especially preferred herein.
- progeny' is understood to embrace both, “asexually” and “sexually” generated progeny of transgenic plants. This definition is also meant to include all mutants and variants obtainable by means of known processes, such as for example cell fusion or mutant selection and which still exhibit the characteristic properties of the initial transformed plant, together with all crossing and fusion products of the transformed plant material.
- Another object of the invention concerns the proliferation material of transgenic plants.
- transgenic plants are defined relative to the invention as any plant material that may be propagated sexually or asexually in vivo or in vitro. Particularly preferred within the scope of the present invention are protoplasts, cells, calli, tissues, organs, seeds, embryos, pollen, egg cells, zygotes, together with any other propagating material obtained from transgenic plants.
- Parts of plants such as for example flowers, stems, fruits, leaves, roots originating in transgenic plants or their progeny previously transformed by means of the process of the invention and therefore consisting at least in part of transgenic cells, are also an object of the present invention.
- the VirD2 is purified by a 4-step procedure to near homogeneity. Initially, the protein is found in the insoluble pellet obtained by highspeed centrifugation of lysed cells. Possibly, upon overproduction of VirD2 in E. coli, the protein is deposited in inclusion bodies. Following extensive washing of the insoluble protein under high salt conditions, VirD2 is solubilized in 6 M urea. Stepwise dialysis against buffers of decreasing urea concentration resulted in a VirD2 fraction soluble under physiological conditions. The following purification steps involved affinity chromatography on heparin-Sepharose and ion-exchange chromatography on DEAE- Sephacel.
- a final gel filtration on Superose 12 using FPLC removed VirD2 degradation products that co-purify on the first two columns, resulting in a 93 % pure VirD2 fraction.
- N-terminal microsequencing of the overproduced and purified VirD2 protein revealed an amino acid sequence that is in agreement with the proposed start of the pTiC58 VirDl gene.
- a solid phase immuno assay using Vir2-specific antiserum revealed identical sizes for VirDl gene products encoded by a gene under its original translational control and those obtained from cells containing pPS 11, the overexpression plasmid.
- the method according to the invention can be advantageously used to increase the transformation efficiency of non-Agrobacterium mediated transformation processes, in that, for example, less transforming DNA is needed as compared to the conventional techniques.
- the qualitiy of the integrated DNA can be improved by the precision of the integration process, and possible rearrangements which are likely to happen to naked DNA can be avoided.
- a reaction batch typically contains about 50 to 500 ⁇ g/ml of DNA in the buffer solution recommended by the manufacturer, New England Biolabs, Beverly, MA. 2 to 5 Units of endonucleases are added for each ⁇ g of DNA and the reaction batch is incubated for from one to three hours at the temperature recommended by the manufacturer. The reaction is terminated by heating at 65 °C for 10 minutes or by extraction with phenol, followed by precipitation of the DNA with ethanol. This technique is also described on pages 104 to 106 of the Maniatis et al (1982) reference.
- DNA polymerase I 50 to 500 ⁇ g/ml of DNA fragments are added to a reaction batch in the buffer recom ⁇ mended by the manufacturer, New England Biolabs.
- the reaction batch contains all four deoxynucleotide triphosphates in concentrations of 0.2 mM.
- the reaction takes place over a period of 30 minutes at 15°C and is then terminated by heating at 65°C for 10 minutes.
- restriction endonucleases such as EcoRI and BamHI
- the large fragment, or Klenow fragment, of DNA polymerase I is used.
- T4 DNA polymerase For fragments obtained by means of endonucleases that produce 3 '-projecting ends, such as PstI and Sad, the T4 DNA polymerase is used. The use of these two enzymes is described on pages 113 to 121 of the Maniatis et al (1982) reference.
- Agarose gel electrophoresis is carried out in a horizontal apparatus, as described on pages 150 to 163 of the Maniatis et al reference.
- the buffer used is the tris-borate buffer described therein.
- the DNA fragments are stained using 0.5 ⁇ g/ml of ethidium bromide which is either present in the gel of tank buffer during electrophoresis or is added after electrophoresis.
- the DNA is made visible by illumination with long- wave ultraviolet light. If the fragments are to be separated from the gel, an agarose is used that gels at low temperature and is obtainable from Sigma Chemical, St. Louis, Missouri.
- the desired fragment is cut out, placed in a plastics test tube, heated at 65 °C for about 15 minutes, extracted three times with phenol and precipitated twice with ethanol. This procedure is slightly different from that described by Maniatis et al (1982) on page 170.
- the DNA can be isolated from the agarose with the aid of the Geneclean kit (Bio 101 Inc., La Jolla, CA, USA).
- the molecule is optionally first treated with DNA-polymerase in order to produce blunt ends, as described in the section above.
- DNA-polymerase in order to produce blunt ends, as described in the section above.
- About 0.1 to 1.0 ⁇ g of this fragment is added to about 10 ng of phosphorylated linker DNA, obtained from New England Biolabs, in a volume of 20 to 30 ⁇ l with 2 ⁇ l of T4 DNA ligase from New England Biolabs, and 1 mM ATP in the buffer recommended by the manufacturer. After incubation overnight at 15C, the reaction is terminated by heating at 65C for 10 minutes.
- the reaction batch is diluted to about 100 ⁇ l in a buffer appropriate for the restriction endonuclease that cleaves the synthetic linker sequence. About 50 to 200 units of this endonuclease are added. The mixture is incubated for 2 to 6 hours at the appropriate temperature, then the fragment is subjected to agarose gel electrophoresis and purified as described above. The resulting fragment will then have ends with endings that were produced by cleaving with the restriction endonuclease. These are usually cohesive, so that the resulting fragment can then readily be linked to other fragments having the same cohesive ends.
- fragments having complementary cohesive ends are to be linked to one another, about 100 ng of each fragment are incubated in a reaction mixture of 20 to 40 ⁇ l containing about 0.2 unit of T4 DNA ligase from New England Biolabs in the buffer recommended by the manufacturer. Incubation is caixied out for 1 to 20 hours at 15 C. If DNA fragments having blunt ends are to be linked, they are incubated as above except that the amount of T4 DNA ligase is increased to 2 to 4 units.
- Escherichia coli strain SCSI obtained from Stratagene, LaJolla, CA
- a high trans ⁇ formation variant of DH1 [Hanahan D, J Mol Biol 166: 557-580 (1983)] is used predominantly as host for plasmids.
- DNA is introduced into E. coli using the calcium chloride method, as described by Maniatis et al (1982), pages 250 and 251 or via electroporation.
- the double-stranded replicative form of the phage M13 derivatives is used for routine processes, such as cleaving with restriction endonuclease, linking etc..
- enzymes can be obtained from Boehringer, New England Biolabs or BRL. They are used in accordance with the manufacturer's instructions unless otherwise indicated.
- Oligo-deoxyribonucleotides are labeled either at their 3'-ends using [ ⁇ - 32 P]ddATP (110 TBq/mmol) and terminal transferase (Amersham) or at their 5'-ends using [ ⁇ - 32 P]ATP (110 TBq/mmol) and phage T4 polynucleotide kinase (31). Standard molecular cloning techniques are performed as described in Sambrook et al [Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab, Cold Spring Harbor, NY (1989).
- the extracted DNA is first treated with restriction enzymes, then subjected to electro ⁇ phoresis in a 0.8 % to 1 % agarose gel, transferred to a nitrocellulose membrane [Southern E.M. (1975)] and hybridized with the DNA to be detected which has previously been subjected to nick-translation (DNA-specific activities of 5 x 10° to 10 x 10° cpm/ ⁇ g).
- the filters are washed three times for 1 hour each time with an aqueous solution of 0.03M sodium citrate and 0.3M sodium chloride at 65C.
- the hybridized DNA is made visible by blackening an X-ray film over a period of 24 to 48 hours.
- VirD2 In order to express to high levels the VirD2 gene [from pTiA6] in E.coli, the reading frame of VirD2 is placed under the control of the strong translation and expression signals of phage T7 gene 10 in plasmid pET3a [Studier et al, in: Methods of Enzymology Vol 185: 60-89 (1990)].
- the VirD2 coding sequence is amplified by Polymerase Chain Reaction [PCR] with primers pl [5'GGGCTCGAGCATATGCCCGATCGCGCTC3 ⁇ SEQ ID NO: 14] and p2 [5'CCCGAGCTCGGATCCCTAGGTCCCCCCGCGCCC3', SEQ ID NO: 15] using plasmid pVD43 [Rossi et al, Mol Gen Genet, (1993)] as template.
- the 1.3 kb reaction product is isolated by gel electrophoresis and inserted in plasmid pTZ19U [commercially available from BioRad] at the Smal site resulting in plasmid pTZ19VirD2.
- the sequence of the 5' end of the gene is confirmed by dideoxy chain termination sequencing, which can be done, for example, by using Sequenase from USB [United States Biochemical] according to the instrucitons provided by the supplier.
- a Ndel-Sall restriction fragment from pTZ19VirD2 is ligated together with a Sall-EcoRI restriction fragment from plasmid pVD43 into plasmid pET3a digested with Ndel and EcoRI giving plasmid pET3aVirD2.
- Recombinant clones are selected on the basis of the presence of the 1.3 kb insert by digestion with Ndel and EcoRI.
- Plasmid pET3aVirD2 is introduced by electroporation into E. coli strain BL21(DE3) [Studier et al, in: Methods of Enzymology Vol 185: 60-89 (1990)]. Cultures are grown in LB medium containing 100 ⁇ g/ml ampicilline at 37°C with shaking. At an optical density of A 600 - ⁇ 0.5-1, expression of VirD2 is induced by the addition of IPTG [Isopropyl ⁇ -D thiogalactopyranoside] to a final concentration of lmM. Shaking is continued for 5 hours. The cells are then pelleted by centrifugation at 4,000xg at 4°C for 30 min. The cell pellet is stored at -80°C until further use.
- VirD2 protein in amounts suitable for biochemical analysis the original translational initiation signal preceding the VirDl structural gene of plasmid pTiC58 is replaced by that of phage T7 gene 10.
- the gene is placed under control of the / c/-regulated tac promoter of expression vector pMS119HE resulting in the VirD2 overproducing plasmid pPSl l.
- IPTG IPTG
- SDS-soluble E. coli cell extracts consisted of VirD2.
- the 1,410 bp SacI-BamHI fragment of pVIR97.89 [Alt-Moerbe et al, (1986) EMBO J. 5, 1129-1135] carrying the VirD2 reading frame except for the first five codons is inserted between the Ndel and BamHI sites of the poly linker sequence of the T7 promoter ⁇ lO / gene 10 SD expression plasmid pT7-7 [described in Sano & Cantor, (1990) Proc. Natl. Acad. Sci. USA 87, 142-146].
- Synthetic oligodeoxyribonucleotides are applied to restore the original 5'-end of the gene and to link the Sad cohesive end of the VirD2 fragment to the Ndel-end of the vector molecule (OL1 : 5'-TATGCCCGATCGAGCT-3'(SEQ ID NO: 36); OL2: 5'-CGATCGGGCA-3' (SEQ ID NO: 37) complementary to part of OLl).
- the manipulated gene is inserted as an Xbal-Hin ⁇ TII fragment in the multi-cloning site of pMS 119HE [Balzer et al, (1992) Nucleic Acids Res. 20, 1851-1858] resulting in pPSll.
- the reading frame of VirD2 is placed under the control of the strong expression signals of phage T7 gene 10 in plasmid pET3a (Studier et al, 1990).
- the VirD2 coding sequence is amplified by Polymerase Chain Reaction with primers
- prl J (5'GGGCTCGAGCATATGCCCGATCGCGCTC3 ', SEQ ID NO: 14) and pr2J
- plasmid pVD43 Rossi et al, 1993
- the 1.3 kb amplified fragment is cloned into the Ndel-EcoRI sites of pET3a resulting in plasmid pFSvirD2.
- Plasmid pFSvirD2 is introduced by electroporation into E. coli strain BL21(DE3) [Studier et al, 1990]. Cultures are grown in LB medium containing 100 ⁇ g ml ampicilline at 37°C with shaking. At A 60 o " 0.5-1, expression is induced by the addition of IPTG [Isopropyl ⁇ -D thiogalactopyranoside] to a final concentration of 1 mM. Shaking is continued for 5 hours. The cells are then pelleted by centrifugation at 4,000xg at 4°C for 30 min.
- Fraction I crude extract: Frozen cells (24.4 g in 120 ml are thawed and lysed by addition of 10 % (w/v) sucrose / 100 mM Tris-HCl, pH 7.6 / 5 M NaCl / 10 %
- the total volume of the lysis mixture is 600 ml. After 1 h at
- Fraction II is applied at a flow rate of 60 ml/h to a DEAE-Sephacel® column (2.6 x 10 cm) equilibrated with buffer B +
- the cells (1 litre culture) are resuspended in 50 ml ice cold lysis buffer [50 mM Tris-HCl pH 8.5, 150 mM NaCl, 5 mM EDTA, 0.1 mM PMSF [phenylmethansulfonylfluorid], 10 mM ⁇ -mercapto ethanol ( ⁇ -ME), 0,5 mg ml lysozyme, 0.1% Brij-35 (polyethylen glycol- dodecylether]. After 1 hour incubation on ice, the lysis mixture is centrifuged at 12,000xg for 30 min at 4°C.
- 50 ml ice cold lysis buffer 50 mM Tris-HCl pH 8.5, 150 mM NaCl, 5 mM EDTA, 0.1 mM PMSF [phenylmethansulfonylfluorid], 10 mM ⁇ -mercapto ethanol ( ⁇ -ME), 0,5 mg ml lys
- the pellet is resuspended in the same volume of lysis buffer without lysozyme, incubated 30 min on ice and centrifuged at 12,000xg for 30 min.
- VirD2 is solubilized by resuspending the pellet in solubilization buffer [50 mM Tris-HCl pH 8.5, ImM EDTA, 0.5 M NaCl, 0.1% Brij-35, 6M urea, 10 mM ⁇ ME].
- the column is washed with 20 ml buffer A and bound proteins are eluted with a linear gradient from 0 to 1 M NaCl in buffer A at a flow rate of 1 ml/min.
- VirD2 elutes at about 400 mM NaCl.
- Fractions [2 ml] containing VirD2 are individually concentrated to 200 ⁇ l by ultrafiltration using, for example, a Centrion 30 device available from Amicon. Glycerol is added to 50% final conentration and fractions are stored at -20°C.
- the cells (1 litre culture) are resuspended in 50 ml ice cold lysis buffer (buffer A) [50 mM Tris-HCl pH 8.5, 150 mM NaCl, 5 mM EDTA, 0.1 mM PMSF (Phenyl-methan- sulfonyl-fluoride), 10 mM ⁇ -mercapto ethanol ( ⁇ -ME), 0.5 mg ml lysozyme, 0.1% Tween-20]. After 1 hour incubation on ice, the lysis mixture is centrifuged at 12,000xg for 30 min at 4°C.
- buffer A buffer
- buffer A 50 mM Tris-HCl pH 8.5, 150 mM NaCl, 5 mM EDTA, 0.1 mM PMSF (Phenyl-methan- sulfonyl-fluoride), 10 mM ⁇ -mercapto ethanol ( ⁇ -ME), 0.5 mg ml lysozyme, 0.
- the pellet is resuspended in the same volume of buffer A without lysozyme, incubated 30 min on ice and centrifuged at 12,000xg for 30 min.
- VirD2 is solubilized by resuspending the pellet in 50 ml buffer B (25 mM NaOAc pH 5, 8M urea, 10 mM ⁇ -ME, 0.1 mM PMSF). After lhour incubation, insoluble material is removed by centrifugation (12,000xg, 45 min) and the supernatant is loaded at a flow rate of 1 ml/min onto an ion exchange column (EconoPac S, BioRad) equilibrated with buffer B.
- ion exchange column EconoPac S, BioRad
- the proteins are eluted with a NaCl gradient (from 0 to 1 M) in buffer B.
- VirD2 peak fractions are pooled and dialysed overnight against 1 liter of buffer C (50 mM Tris-HCl pH 8.5, 0.15 M NaCl, 10 mM ⁇ -ME, 0.05% Tween-20, O.lmM PMSF, 5mM MgCl 2 ).
- Precipitated material is removed by centrifugation [12-000xg, 30 min] and the supernatant diluted two fold and applied to a heparin column [BioRad] equilibrated with buffer C.
- the protein solution is applied at a flow rate of 2 ml/min.
- the column is washed with 20 ml buffer C and bound proteins are eluted with a linear gradient from 0.15 to 1 M NaCl in buffer C at a flow rate of 1 ml/min.
- VirD2 peak fraction eluting at about 400 mM NaCl, is dialysed overnight against buffer C. Insoluble material is removed by centrifugation [12,000xg, 30 min]. Aliquots are supplemented with 10% glycerol, frozen on dry ice and stored at -80°C.
- the heparin-purified protein is further purified on reverse-phase HPLC column as follows:
- VirD2 samples (lOO ⁇ g, 200 ⁇ l) are injected onto a C18 reverse phase column (Vydac) equilibrated with 0.1% trifluoroacetic acid [TFA].
- TFA trifluoroacetic acid
- the bound protein is eluted (flow rate 0.5ml/min) with a linear acetonitrile gradient (from 0 to 70% in 1 hour) in 0.1% TFA.
- the VirD2 peak fraction, eluting at 48% acetonitrile is lyophilised in a SpeedVac apparatus.
- VirD2 is resuspended in buffer D [20mM Tris-HCl, pH 8.5, 5mM MgCl 2 , 50mM NaCl, 0.05% Tween-20, 10% (v/v) glycerol], frozen on dry ice and stored at -80°C.
- Vir D2 containing fractions are checked for activity using the oligonucleotide cleavage assay descirbed in Pansegrau et al [Proc Nat Acad Sci USA, (1993b)].
- a 17-mer oligonucleotide homologous to the pTiA6 right border sequence [5'GGTATATATCC- TGCCAG3', SEQ ID NO: 17] is used as a substrate.
- the 13-mer reaction product is analyzed by electrophoresis on 19% acrylamide gels containing 8 M urea.
- Reaction products are quantified by autoradiography of gels with the storage phosphor technology [Johnston et al, Electrophoresis 1_1: 355-360 (1990)] Where appropriate, the cleavage reaction is followed by trypsin digestion in presence of 0.01 % SDS.
- the following oligonucleotides are used as substrates: pTiC58 RB (right border), d(p*CCAATATATCCTG V TCAA) (SEQ ID NO: 18); pTiA6 RB, d(p*GGTATATATCCTG V CCAG) (SEQ ID NO: 19); RP4 oriT, d(p*TTCACCTATCCTG V CCCG) (SEQ ID NO: 20). Positions of the cleavage sites are indicated by wedges. The radioactive label is symbolized by an asterisk.
- Occurrence of a 13-mer indicates that VirD2 mediates cleavage at the same site within the pTiC58 border sequences that is found by analysis of T-DNA produced in vivo [D ⁇ rrenberger et al, Proc. Natl. Acad. Sci. USA 86, 9154-9158 (1989)]. Cleavage at an equivalent site is also found using a VirD2 protein obtained from pTiA6.
- Example 3 VirD2 covalently attaches to the 5' -end of cleaved oligonucleotides.
- VirD2 has been found to be covalently associated with the 5 '-ends of the T-DNA single strands that are produced in agrobacteria induced by plant phenolic compounds like acetosyringone.
- 3 '-labeled oligonucleotide is incubated in presence of Mg 2+ ions with VirD2 and the products are separated on a sequencing gel.
- Oligonucleotide cleavage under these conditions resulted in a labeled species that did not enter the sequencing gel, indicating that the 3 '-terminal oligonucleotide moiety is tightly associated with high molecular weight material
- Treatment of the material with a variety of proteases resulted in labeled species with distinct electrophoretic mobilities reflecting the covalent association of the 5 '-end of cleaved oligonucleotides with different peptide species generated by proteases of different specificity
- Example 4 VirD2 transfers a covalently attached oligonucleotide moiety to a preformed border sequence 3'-end
- a preformed border 3 '-terminus is synthesized as an 13-mer oligonucleotide and incubated in various amounts together with VirD2 protein and a 3 '-end labeled 30-mer oligonucleotide carrying the pTiC58 right border cleavage site.
- Specific transfer of the 3 '-terminal moiety of the 30-mer to the 13-mer by VirD2 resulted in a 22-mer detectable on polyacrylamide gels by the 3 '-label.
- Example 5 Site specific Cleavage of large Oligonucleotides bv VirD2 Ml 3 phages are constructed that contain the pTi plasmid border sequence and single-stranded substrates longer than the oligonucleotides reported in Example 2. The procedure is the following:
- PCR fragment containing as well the border sequence is amplified using a pair of primers (UP-40: 5'GTTTTCCCAGTCACGAC3' (SEQ ID NO: 22) and UP-112: 5'CACTC- ATTAGGCACCCCAGGC3' (SEQ ID NO: 23)).
- the PCR product is 249 bp long.
- the DNA is gel purified, phenol extracted and precipitated. Two to 4 % of this dsDNA are used in a second amplification step using only the UP-40 primer.
- the next amplification step (40 cycles) is linear and produces single-stranded molecules corresponding to only one of the strands of the input dsDNA.
- This single-stranded DNA is expected to be processed by VirD2.
- the ssDNA product is gel purified, phenol extracted and precipitated. Hundred ng are labeled with ⁇ 32 P-ATP and polynucleotide kinase using the supplier's conditions. After the labeling reaction, the DNA is phenol extracted, precipitated and resuspended in 100 ⁇ l of water.
- the in vitro cleavage is performed in the conditions previously described, using 1 ng ssDNA per assay.
- a kinetic analysis revealed that a few minutes incubation of the VirD2 preparation with the substrate produces a well defined band, shorter than the input DNA (as expected).
- VirD2 substrates are single stranded DNA molecules containing the pTiA6 Right Border sequence.
- Two complementary oligonucleotides top strand : 5'AATTCTGGCAGG- ATATATACCGTTGTAATTTGTAC3' (SEQ ID NO: 24) and bottom strand : 5'AA- ATTACAACGGTATATATCCTGCCAG3' (SEQ ID NO: 25)) are annealed and ligated into pTZ19U digested with EcoRI and Kpnl to produce pZ19URB.
- a EcoRI fragment from pGUS23 [Puchta et al, Mol Cell Biol 12: 3372-3379 (1992)] containing the GUS gene is introduced into pZ19URB to produce pZ19URBGUS.
- a EcoRI/Hindlll fragment from pZ19URBGUS is subcloned into M13mpl8 and M13mpl9 to obtain pM18RBGUS and pM19RBGUS respectively.
- Control Ml 3 vectors are constructed by insertion of the EcoRI fragment of plasmid pGUS23 into M13mpl8 cleaved at the EcoRI site giving pM18GUS(+) (GUS coding strand is present in the viral (+) strand) and pM18GUS(-) (GUS coding strand is present in the viral (-) strand). These two vectors lack the pTiA6 border sequence. Single stranded DNA is prepared as described in [Sambrook et al, (1989)] and are further purified using a Qiagen DNA [QIAGEN Inc., CA, USA] purification column according to the manufacturer instructions.
- the VirD2 protein is allowed to react with single stranded DNA vector containing the pTiA6 right border sequence, using the vectors without the border sequence as controls. Reaction conditions are similar to those described in Pansegrau et al (1993b). Reaction buffer is 20 mM Tris-HCl, pH 8.5, 5 mM MgCl 2 , 1 mM EDTA, 50 mM NaCl. Ten pmole VirD2 are reacted with 1 pmole single-stranded DNA substrate. Cleavage is allowed to proceed for one hour at 37°C Upon cleavage, the protein becomes linked to the 5' end of the DNA that is thereby linearized.
- Quantification of the ssDNA cleavage is checked by 3' end labelling aliquots of the reaction mixture with Terminal Transferase (Boehringer) using a- ⁇ P-ddATP as label. Incorporation of the * ⁇ 2 P label in ethanol-precipitable is a measure of the extent of ssDNA cleavage by VirD2.
- Free VirD2 is removed from the reaction mixture by repeated dilution with reaction buffer and ultrafiltration with an Centricon 100 device.
- VirD2 substrates are single stranded DNA molecules containing the pTiA6 Right Border and overdrive sequence (Peralta et al, 1986).
- a 108 bp EcoRI-Sall fragment from pTD20 (5 'aattccagcTGGCAGGATATATACCGTTGTAATTTgagctcgtgtg aaTAAGTCGCTGT- GTATGTTTGTTTGattgcggccgcaagctttctagaggatccctgcagg 3', SEQ ED NO: 26) (Right border and Overdrive sequence are in upper case letters) is cloned into the EcoRI-Sall sites of M13mpl9 resulting in pM13RB.
- a further plasmid (pM13PstGUS) is constructed, which is almost identical to pM13RBGUS, except that the GUS gene, oriented in the opposite direction, is flanked by two PstI sites.
- This vector can be used to quantify the extent of VirD2 processing using a primer extension technique (see below). It can also be used to produce linear single-stranded DNA by digestion with the PstI restriction endonuclease.
- the transformation is carried out with a spray-type particle gun a described in EP-A 434,616.
- the target tissue used are tobacco SRI leave pieces of 0.5 to 2.0 cm.
- the plant material is preferably submitted to plasmolysis [20% maltose treatment] before shooting.
- Single-stranded DNA is prepared as described in (Sambrook et al, 1989) and further purified by CsCl gradient centrifugation.
- the VirD2 fractions are checked for activity using the oligonucleotide cleavage assay described in Pansegrau et al (1993b). For example, a 17-mer oligonucleotide homologous to the pTiA6 right border sequence (5'GGTATATATCCTGCCAG3 ⁇ SEQ ID NO: 27) is used as substrate. The 13-mer cleavage products are analysed by electrophoresis on 20% acrylamide gels containing 8 M urea.
- the VirD2 protein is allowed to react with single-stranded DNA containing the pTLA6 right border sequence, using vectors lacking the border sequence as controls.
- reaction 5 micrograms of purified VirD2 are allowed to react with 1 microgram of single-stranded DNA. Reaction (20 ⁇ l) is performed in the standard reaction buffer (20mM Tris-HCl, pH 8.5, 5mM MgCl 2 , 50mM NaCl) at 37°C for 15 minutes. The reaction mixture is used as such either immediately or is frozen on dry ice and thawed immediately before use.
- Quantification of the ss DNA cleavage is done by performing a primer extension reaction, using a linear ss DNA (obtained by digestion of pM13PstGUS with PstI). After the reaction of VirD2 with its linear ssDNA substrate is completed, the reaction mixture is digested wid Proteinase K, in order to remove the protein covalently linked to the 5' end of the cutting site. After phenol extraction, the DNA is precipitated with ethanol and used in a primer extension reaction using the Sequenase kit (USB) according to the instructions concerning the sequencing of single-stranded templates. The primer used is located about 100 nucleotides 3' from the VirD2 cutting site.
- the labelling reaction ( ⁇ - 35 S)-labelled dATP and unlabelled dCTP, dGTP and dTTP.
- the labelling reaction (the extension) is allowed to proceed at 37°C for 10 minutes.
- the extension reaction is terminated either at the cut introduced by VirD2 or at the extremity of the linear molecule in case VirD2 did not process it. Therefore the ratio between prematurely terminated extension products and full length products is a measure of the extent of cutting by VirD2.
- An normal sequencing reaction is performed in parallel on non-processed DNA with the same primer. One can then identify the cut site by comparison with the sequencing ladder.
- Free VirD2 can be removed from the reaction mixture by repeated dilution with reaction buffer and ultrafiltration with an Centricon 100 device.
- VirDl To allow effective translational initiation in E. coli the original SD sequence preceding the VirDl gene is replaced by that of phage T7 gene 10. Two CGG triplets of a cluster of rare Arg codons are altered by site directed mutagenesis. This prevents termination of translation at this position and thus production of truncated VirDl molecules.
- the modified VirDl gene is placed under control of the Lacl-regulated tac promotor in the expression vector pMS119HE resulting in the VirDl overproducing plasmid pPS20. Following chemical induction of gene expression by IPTG addition to the culture medium approx. 8 % of SDS-soluble cell protein consists of VirDl.
- the rare CGG Arg codons are changed by site directed mutagenesis following the procedure of Sayers et al. (1988).
- the sequence CGG CGG CGG (nucleotides 1048-1056 of Atuvird, accession number M33673) is mutated to CGC CGG CGT.
- the manipulated VirDl gene with the changed codons and the T7 gene 10 Shine-Dalgano sequence is inserted as an Xbal-SacI fragment in the multi-cloning site of pMS119HE [Balzer et al., 1992] resulting in pPS20.
- Cells are harvested by centrifugation (16,000 x g, 10 min), resuspended in 0.1 M spermidine tris(hydrochloride) / 0.2 M NaCl / 2 mM EDTA / pH 7.6 to a final concentration of 150 Asoo/ml and frozen in liquid nitrogen. All subsequent procedures are performed at 0-4° C, contact of the protein solution to metal surfaces is strictly avoided.
- Fraction I crude extract. Frozen cells (29 g in 90 ml) are thawed and lysed in 4.5 % (wt/vol) sucrose / 70 mM Tris-HCl (pH 7.6) / 1 M NaCl / 0.25 % (wt/vol) Brij-58 / 0.75 mg/ml lysozyme. The total volume of the lysis mixture is 460 ml. After 1 hr incubation at 0° C the inclusion bodies, containing the VirDl protein, are pelleted by centrifugation (100,000 x g, 1 hr).
- pellets are washed thoroughly with 3 M urea / 1 M NaCl / 1 mM EDTA / 0.5 mg/ml sodium deoxycholate. This washing step is repeated.
- pellets are eluted with 150 ml buffer A [20 mM Tris-HCl (pH 8.7) / 500 mM NaCl / 1 mM EDTA / 0.05 % (wt/vol) Brij-58 / 10 % (wt vol) glycerol] / 8 M urea / 10 mM DTT overnight and centrifuged (100,000 x g, 1 hr).
- the protein solution is dialyzed for 6 hr each against six changes of buffer A (2 liter) containing 5, 4, 3, 2, 1, 0.5 M urea then against four changes of buffer B [20 mM Tris-HCl (pH7.6) / 1 mM EDTA / 1 mM DTT / 0.01 % (wt/vol) Brij-58 / 10 % (wt vol) glycerol] (2 liter) containing 250, 125, 65, 0 mM NaCl.
- the dialyzed solution is centrifuged (100,000 x g) and the pellet discarded (fraction I, 240 ⁇ ). Fraction II, DEAE-Sephacel.
- Fraction I is loaded on a DEAE-Sephacel column (2.6 x 20 cm) equilibrated with buffer B at a flow rate of 54 ml/hr. The column is washed with 200 ml buffer B and the VirDl containing fractions of the flowthrough are pooled (fraction II, 360 ml).
- Fraction HI phosphocellulose-Pl l.
- Fraction II is applied at a flow rate of 55 ml/hr to a phosphocellulose Pl l column (2.6 x 24 cm) equilibrated with buffer C [30 mM Tris-phosphate (pH 7.0) / 1 mM EDTA / 1 mM DTT / 10 % (wt/vol) glycerol].
- the column is washed with 150 ml of buffer C and 50 ml of buffer C / 50 mM NaCl.
- proteins are eluted with a 1.4-1 gradient from 50 to 750 mM NaCl in buffer C.
- VirDl elutes at about 375 mM NaCl.
- Example 8 Specific : cleavage reaction of forml DNA by VirDl and VirD2 in vitro.
- Substrates pPSlOO form I DNA pBR329 ⁇ [BamHI-Sall, right T border of pTiC58, 43 bp], 3918 bp
- pPS 101 form I DNA pPSlOO ⁇ [Pstl-Aatll, left T border of pTiC58, 43 bp], 3284 bp
- pPSl 10 form I DNA pPSlOO ⁇ [Nhel-BamHI, overdrive of pTiA6, 40 bp], 3812 bp
- pPSl 11 form I DNA pPSlOl ⁇ [Nhel-BamHI, overdrive of pTiA6, 40 bp], 3178 bp
- pS 124.1 form I DNA containing two right borders and one overdrive element of pTiA6 [Alt-Moerbe etal, (1990)]
- Form I substrate DNA (0.7 ⁇ g) is incubated with VirDl (750 ng, 47 pmol) and VirD2 (550 ng, 11 pmol) in a total volume of 20 ⁇ l TNM (20 mM Tris-HCl (pH 8.8) / 50 mM NaCl / 5 mM MgCl 2 ), for 40 min at 37 C. Cleavage products are analyzed on vertical 0.7 % agarose gels after proteinase K digestion in the presence of 1 % SDS. The cleavage reaction requires form I DNA, is T border specific, VirDl-, VirD2- and Mg 2+ -dependent.
- Reaction products are quantified by scanning with a Fluorlmager 575 (Molecular Dynamics).
- the yield of specifically cleaved DNA is 35-95 % of the input form I DNA, depending on the substrate.
- Substrates pPSlOO and pPSHO containing one border element are cleaved to 35 %, pPSlOl and pPSl l l to 70 %, pS 124.1 to 95 %.
- the higher yield of cleaved plasmid DNA from substrates carrying two border sequences is due to a higher probability for cleavage to occur at one of the nick-sites.
- Specificity and nick-site were determined by electrophoresis of linearized reaction products on alkaline agarose gels and runoff sequencing.
- cleavage site is identical to that found in vivo [Diirrenberger et al, (1989)] or with single-stranded DNA substrates and VirD2 in vitro [Pansegrau et al., (1993)]. Likewise, cleavage results in covalent attachment of VirD2 protein to the 5-terminus of the broken DNA strand.
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
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- MOLECULE TYPE DNA (genomic)
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- HYPOTHETICAL NO
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- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic) ⁇
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- MOLECULE TYPE DNA (genomic)
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- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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Abstract
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Priority Applications (1)
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AU75345/94A AU7534594A (en) | 1993-08-13 | 1994-08-03 | Method for stable transformation of plants |
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EP93113028 | 1993-08-13 | ||
EP93113028.0 | 1993-08-13 |
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WO1995005471A2 true WO1995005471A2 (en) | 1995-02-23 |
WO1995005471A3 WO1995005471A3 (en) | 1995-06-01 |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997012046A1 (en) * | 1995-09-25 | 1997-04-03 | Novartis Ag | Improved integration of exogenous dna delivered to eukaryotic cells |
WO1999061619A2 (en) * | 1998-05-22 | 1999-12-02 | Pioneer Hi-Bred International, Inc. | Cell cycle genes, proteins and uses thereof |
WO2000005392A1 (en) * | 1998-07-24 | 2000-02-03 | Novartis Ag | Method for transformation of animal cells |
US6051409A (en) * | 1995-09-25 | 2000-04-18 | Novartis Finance Corporation | Method for achieving integration of exogenous DNA delivered by non-biological means to plant cells |
WO2004020641A1 (en) * | 2002-09-02 | 2004-03-11 | Dalian Keyuan Agricultural Bioengineering Co., Ltd. | Plant gene engineering manipulation method with biological safety |
WO2005026371A1 (en) * | 2003-09-09 | 2005-03-24 | Osaka University | Method of transferring biological substance and transformation method using the method of transferring biological substance |
US7928291B2 (en) | 2006-07-19 | 2011-04-19 | Monsanto Technology Llc | Use of multiple transformation enhancer sequences to improve plant transformation efficiency |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989009776A1 (en) * | 1988-04-14 | 1989-10-19 | Regents Of The University Of Minnesota | VirE OPERON-ENCODED POLYPEPTIDES THAT BIND SINGLE-STRANDED DNA |
-
1994
- 1994-08-03 WO PCT/EP1994/002566 patent/WO1995005471A2/en active Application Filing
- 1994-08-03 AU AU75345/94A patent/AU7534594A/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989009776A1 (en) * | 1988-04-14 | 1989-10-19 | Regents Of The University Of Minnesota | VirE OPERON-ENCODED POLYPEPTIDES THAT BIND SINGLE-STRANDED DNA |
Non-Patent Citations (11)
Title |
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BIOLOGICAL ABSTRACTS, vol. 89 1990, Philadelphia, PA, US; abstract no. 104025, SRINIVASAN, M.F.Y., ET AL. '25-bp long border sequence of T-DNA is sufficient for cleavage by vird endonuclease of Agrobacterium' & J. GENET., vol.68, no.3, 1989 pages 161 - 170 * |
DATABASE BIOSIS BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US DN 97358502 VOGEL, A.M., ET AL. 'Mutational analysis of Agrobacterium tumefaciens pTiA6 virD1: identification of functionally important residues' & MOLECULAR MICROBIOLOGY, vol.12, no.5, 1994 pages 811 - 817 * |
EMBO JOURNAL, vol.7, no.13, 1988, EYNSHAM, OXFORD GB pages 4055 - 4062 HERREA-ESTRELLA, A., ET AL. 'VirD proteins of Agrobacterium tumefaciens are required for the formation of a covalent DNA - protein complex at the 5' terminus of T-strand molecules' * |
J. BACTERIOLOGY, vol.174, no.1, January 1992 pages 303 - 308 VOGEL, A.M., ET AL. 'Mutational analysis of Agrobacterium tunefaciens virD2: tyrosine 29 is essential for endonuclease activity' * |
MOLECULAR AND GENERAL GENETICS, vol.239, no.3, June 1993, BERLIN DE pages 345 - 353 ROSSI, L., ET AL. 'The VirD2 protein of Agrobacterium tumefaciens carries nuclear localization signals important for transfer of T-DNA to plants' * |
MOLECULAR MICROBIOLOGY, vol.8, no.5, May 1993 pages 915 - 926 FILICHKIN, S.A., ET AL. 'Formation of a putative relaxation intermediate during T-DNA processing directed by the Agrobacterium tumefaciens VirD1,D2 endonuclease' * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol.86, May 1989, WASHINGTON US pages 3109 - 3113 GHAI, J., ET AL. 'The VirD operon of Agrobacterium tumefaciens Ti plasmid encodes a DNA relaxing enzyme' * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol.86, no.11, June 1989, WASHINGTON US pages 4017 - 4021 HOWARD, E.A., ET AL. 'Activation of the T-DNA transfer process in Agrobacterium results in the generation of a T-strand-protein complex: Tight association of virD2 with the 5' ends of T-strands' * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol.90, April 1993, WASHINGTON US pages 2925 - 2929 PANSEGRAU, W., ET AL. 'Relaxase (TraI) of IncPalpha plasmid RP4 catalyzes a site-specific cleaving-joining reaction of single-stranded DNA' * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol.90, December 1993, WASHINGTON US pages 11538 - 11642 PANSEGRAU, W., ET AL. 'Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefaciens Ti plasmids: Analogy to bacterial conjugation' * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol.91, January 1994, WASHINGTON US pages 694 - 698 JASPER, F., ET AL. 'Agrobacterium T-strand production in vitro: Sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein' * |
Cited By (17)
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KR100477413B1 (en) * | 1995-09-25 | 2006-01-27 | 신젠타 파티서페이션즈 아게 | Improved integration of exogenous DNA delivered to eukaryotic cells |
AU707948B2 (en) * | 1995-09-25 | 1999-07-22 | Syngenta Participations Ag | Improved integration of exogenous DNA delivered to eukaryotic cells |
US6051409A (en) * | 1995-09-25 | 2000-04-18 | Novartis Finance Corporation | Method for achieving integration of exogenous DNA delivered by non-biological means to plant cells |
US6410329B1 (en) | 1995-09-25 | 2002-06-25 | Novartis Finance Corporation | Method for achieving site specific integration of exogenous DNA delivered by non-biological means to plant cells |
WO1997012046A1 (en) * | 1995-09-25 | 1997-04-03 | Novartis Ag | Improved integration of exogenous dna delivered to eukaryotic cells |
WO1999061619A2 (en) * | 1998-05-22 | 1999-12-02 | Pioneer Hi-Bred International, Inc. | Cell cycle genes, proteins and uses thereof |
WO1999061619A3 (en) * | 1998-05-22 | 2000-03-23 | Pioneer Hi Bred Int | Cell cycle genes, proteins and uses thereof |
WO2000005392A1 (en) * | 1998-07-24 | 2000-02-03 | Novartis Ag | Method for transformation of animal cells |
US6498011B2 (en) | 1998-07-24 | 2002-12-24 | Novartis Ag | Method for transformation of animal cells |
WO2004020641A1 (en) * | 2002-09-02 | 2004-03-11 | Dalian Keyuan Agricultural Bioengineering Co., Ltd. | Plant gene engineering manipulation method with biological safety |
WO2005026371A1 (en) * | 2003-09-09 | 2005-03-24 | Osaka University | Method of transferring biological substance and transformation method using the method of transferring biological substance |
US7928291B2 (en) | 2006-07-19 | 2011-04-19 | Monsanto Technology Llc | Use of multiple transformation enhancer sequences to improve plant transformation efficiency |
US8404931B2 (en) | 2006-07-19 | 2013-03-26 | Monsanto Technology Llc | Use of multiple transformation enhancer sequences to improve plant transformation efficiency |
US8759616B2 (en) | 2006-07-19 | 2014-06-24 | Monsanto Technology Llc | Use of multiple transformation enhancer sequences to improve plant transformation efficiency |
US9464295B2 (en) | 2006-07-19 | 2016-10-11 | Monsanto Technology Llc | Use of multiple transformation enhancer sequences to improve plant transformation efficiency |
US10066235B2 (en) | 2006-07-19 | 2018-09-04 | Monsanto Technology Llc | Use of multiple transformation enhancer sequences to improve plant transformation efficiency |
US10865418B2 (en) | 2006-07-19 | 2020-12-15 | Monsanto Technology Llc | Use of multiple transformation enhancer sequences to improve plant transformation efficiency |
Also Published As
Publication number | Publication date |
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AU7534594A (en) | 1995-03-14 |
WO1995005471A3 (en) | 1995-06-01 |
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